Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada. simpson@cmmt.ubc.ca.

Abstract

BACKGROUND:

The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo.

RESULTS:

In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear.

CONCLUSIONS:

We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

Human bacterial artificial chromosomes can be targeted at Hprt by homologous recombination and, if desired, conditionally removed using cre recombinase. (a) Integration into the mouse genome of the bacterial artificial chromosome (BAC)-lacZ-reporter constructs by homologous recombination results in the human gene in either direction relative to the X chromosome; this schematic presents one possible orientation. Regardless of orientation, each insertion resulted in the presence of four loxP sites in the genome (two wild-type and one 511 mutant at one end and one wild-type at the other end of the BAC insert). (b) Crossing the BAC-lacZ-reporter females to ACTB-cre males should result in the generation of two different male offspring; BAC-lacZ-reporter animals, wild-type for the ACTB-cre transgene; and BAC-lacZ-reporter animals carrying the ACTB-cre transgene. Only the reporter animals that are positive for the ACTB-cre gene should recombine the outer most loxP sites, resulting in excision of the BAC construct from the genome and leaving one loxP site. This would result in an absence of lacZ-positive signal. hP, human HPRT promoter; h1, human first exon; m2 and m3, mouse second and third exons; mouse homology arms (dark blue); Hprt coding regions (red); vector backbone (yellow with black edges); SacB gene from BAC vector backbone (brown); 5′ and 3′ untranslated regions of the human gene (orange); coding region of the human gene (green); lacZ reporter gene (light blue). Schematic, not to scale. (c-f)lacZ expression results from AMOTL1-lacZ, MAOA-lacZ, NOV-lacZ, and NR2F2-lacZ females bred to the ACTB-cre males are presented. lacZ-positive staining (blue) was detected in AMOTL1-lacZ, MAOA-lacZ, NOV-lacZ, and NR2F2-lacZ males not carrying the ACTB-cre allele whereas absence of staining was detected in males positive for ACTB-cre by genotyping (AMOTL1-lacZ, ACTB-cre; MAOA-lacZ, ACTB-cre; NOV-lacZ, ACTB-cre; NR2F2-lacZ, ACTB-cre), suggesting whole BAC excision from the genome. Scale bar: (c-f) 1 mm. N = 3 animals for all genotypes.

Comparative genomics delineated the DNA boundaries that were sufficient for adult brain-specific expression of AMOTL1 and MAOA. Coordinates corresponding to the human bacterial artificial chromosome (BAC) constructs used in this study were retrieved and visualized using the University of California Santa Cruz (UCSC) genome browser. (a) DNA alignment of the human AMOTL1 BAC (RP11-936P10) with the mouse genome delineated the genomic DNA boundaries sufficient for proper expression of this human gene in the anterior thalamic nuclei in both developing (P7) and adult mouse brain, regions for which this gene was chosen. (b) DNA alignment of the human MAOA BAC (RP11-475M12) with the mouse genome delineated the genomic DNA boundaries sufficient for proper expression of this human gene in the locus coeruleus (LC) in both developing (P7) and adult mouse brain, regions for which this gene was chosen. One hypothesis suggested by our results was that additional conserved regulatory elements in the 3′ coding and non-coding regions (red rectangle) could be important in narrowing the brain expression of this human gene.

Comparative genomics delineated the DNA boundaries that were sufficient for adult brain-specific expression of NOV and NR2F2. Coordinates corresponding to the human bacterial artificial chromosome (BAC) constructs used in this study were retrieved and visualized using the University of California Santa Cruz (UCSC) genome browser. (a) DNA alignment of the human NOV BAC (RP11-840I14) (black), against both the RP23-235B13 BAC construct used in the Gene Expression Nervous System Atlas (GENSAT) mouse model (blue), and the mouse genome, delineated the genomic DNA boundaries sufficient for proper expression of this human gene in the basolateral amygdaloid nuclei, cortical layers, and pyramidal neurons in the cornu ammonis 1 (CA1) regions in the adult brain. One hypothesis suggested by our results was that additional functionally conserved regulatory elements homologous to the large non-overlapping 3′ mouse-BAC region are necessary for proper human-gene expression in the developing cortical layers at P7. (b) DNA alignment of the human NR2F2 BAC (RP11-134D15) (black), against both the RP23-109L9 BAC construct used in the GENSAT mouse model (blue) and the mouse genome, delineated the genomic DNA boundaries sufficient for region-specific expression of this human gene in the basolateral, and corticolateral amygdaloid nuclei in the adult brain. One hypothesis suggested by our results was that additional functionally-conserved regulatory elements homologous to the non-overlapping 5′ mouse-BAC region are necessary for proper expression in the developing hypothalamus at P7. Black rectangle box in (b) is shown in (c). (c) Sequence alignment using the coordinates of the primers used in the BAC lacZ retrofitting process (grey bars) and the cDNA sequence used to generate an anti-NR2F2 antibody (black bar), suggested that the absence of expression of the NR2F2-lacZ constructs in retinal amacrine cells was not attributable to detection of different isoforms of NR2F2.